Tumor necrosis factor α, and agonist and antagonists of cannabinoid receptor type 1 and type 2 alter the immunophenotype of stem cells from human exfoliated deciduous teeth

ABSTRACT Objective To verify the involvement of the endocannabinoid system in the immunomodulatory profile of stem cells from human exfoliated deciduous teeth, in the presence or absence of TNF-α, and agonist and antagonists of CB1 and CB2. Methods Stem cells from human exfoliated deciduous teeth were cultured in the presence or absence of an agonist, anandamide, and two antagonists, AM251 and SR144528, of CB1 and CB2 receptors, with or without TNF-α stimulation. For analysis of immunomodulation, surface molecules linked to immunomodulation, namely human leukocyte antigen-DR isotype (HLA-DR), and programmed death ligands 1 (PD-L1) and 2 (PD-L2) were measured using flow cytometry. Results The inhibition of endocannabinoid receptors together with the proinflammatory effect of TNF-α resulted in increased HLA-DR expression in stem cells from human exfoliated deciduous teeth, as well as, in these cells acquiring an anti-inflammatory profile by enhancing the expression of PD-L1 and PD-L2. Conclusion Stem cells from human exfoliated deciduous teeth respond to the endocannabinoid system and TNF-α by altering key immune response molecules.


❚ METHODS Isolation of SHED
Stem cells from human exfoliated deciduous teeth (SHED) were isolated from a healthy volunteer and cultured according to Gronthos et al. (1) and Liu et al., (51) with some modifications.The dental pulp was dilacerated with a scalpel and then washed in Dulbecco's modified Eagle medium: Nutrient mixture F-12 (DMEM/F12) (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS; LGC Biotecnologia, Brasil), 100U/mL penicillin, 100µg/mL streptomycin, 2mM L-glutamine, and 0.01mM non-essential amino acids (Invitrogen), until the release of cells.The isolated SHED were kept in 75cm² culture flasks in supplemented DMEM/F12 at 37 o C under 5% CO 2 and expanded until 70% confluence.
The acquisition of teeth followed a declaration of consent signed by persons responsible for the minor.All proceedings were performed in accordance with the ethical standards of the local ethics committee (CAAE: 33905014.4.0000.5133;#003/2011).The samples were stored in the Biobank Human Genetics and Cell Therapy (CONEP B-030), Universidade Federal de Juiz de Fora.

Molecular characterization of SHED
The expression of hematopoietic (CD34 and CD45), mesenchymal (NES and CD105), and embryonic (NANOG and OCT4) markers on SHED was analyzed using RT-PCR.Briefly, total RNA was extracted from cells using the RNeasy Mini kit (Qiagen, Dusseldorf, Germany), according to the manufacturer's instructions.RNA quality was determined by concentration and purity of RNA by measuring absorbance at 260 and 280nm.Expression of the genes was analyzed on cDNA fragments obtained with the QIAGEN OneStep RT-PCR Kit (Qiagen) using the primers described in table 1.The products were electrophoresed on 2% agarose gels, stained with ethidium bromide, and visualized with the Gel Logic 100 Imaging System transilluminator (Kodak, New Haven, USA), using Kodak 1D Software, v. 3.6.5K2 (Kodak).The experiment was performed in triplicates.

Osteogenic differentiation of SHED
The differentiation capacity of SHED was assessed in vitro.The established lines were subjected to osteogenic differentiation.The cells were plated in 6-well plates at a density of 1 × 10 4 cells/well.After 24 hours of culture in regular medium, the cells were washed, the medium in the wells was replaced with an osteogenic differentiation medium (DMEM-LG, 10% FBS, 1 × 10 -8 M dexamethasone, 0.2 M L-ascorbic acid 2-phosphate, and 10mM β-glycerol phosphate), and the cells were maintained in culture for 14 days.All experiments were done in triplicates.At the end of the culture period, calcium deposition was evaluated using Alizarin Red S (Sigma-Aldrich, USA) staining, according to the manufacturer's instructions.Images were obtained with an inverted microscope (Nikon TS100F, Japan) using 4× and 10× objective lens (Nikon, Japan).
Images were captured with a camera (TV Lens C-0.6X Nikon, Japan) and acquired with Motic Images Plus 3.0 Routine Software Series.

Cell culture and stimulation of the endocannabinoid system
4)

Assessment of cell viability
Cell (500 cells/well) were plated in 96-well plates, and cultured in the presence of 5µM of anandamide, in the presence or absence of 2µM of AM251 and 2µM of SR144528, with or without TNF-α (10ng/mL) stimulation (Table 2), in supplemented DMEM/F12 at 37 o C under 5% CO 2 .After 48 hours, 10% of 0.15mg/mL resazurin sodium salt (Alamar Blue ® , Thermo Fisher Scientific, USA) was added to the wells.The cells were further incubated for 4 hours and the absorbance was read at 570 and 600nm.The experiment was performed in triplicates.

Flowcytometry analysis of cell markers
For flow cytometry analysis, cultured SHED (Table 2) were washed three times with 1X PBS and enzymatically disaggregated using 0.25% trypsin (TrypLE Express, Invitrogen, USA) for five minutes at 37°C; the enzyme was inactivated by adding the culture medium.The cells were centrifuged at 300 × g for five minutes, counted using a hemocytometer (BD Biosciences) with blue, red, and violet laser, and labeled for the surface markers HLA-DR (V500 mouse anti-human HLA-DR; BD Bioscience), PDL-1 [BB515 mouse anti-human PDL-1, isotype: mouse (BALB/c) IgG1, clone: MIH1; BD Bioscience], and PDL-2 (APC mouse anti-human PDL-2, isotype: mouse IgG1, κ, clone: MIH18; BD Bioscience).A total of 50,000 events were acquired for each group and SHED not labeled with antibodies were used as negative control.The experiment was performed in triplicates.

Statistical analysis
The data collected in vitro were analyzed with appropriate statistical tools taking into account the sample number and normal distribution, using the Shapiro-Wilk normality test.Comparisons between the means of the analyzed groups were made using the Kruskal-Wallis test, and post-hoc Dunn's multiple comparison test.The differences were considered significant at p<0.05 for the two-tailed analysis.Statistical analyses and graphical representation of data were done using the GraphPad Prism software (version 7; GraphPad Software, Inc., USA).

❚ RESULTS Isolation, characterization, and differentiation of SHED
In basal medium, SHED showed a typical fibroblastlike appearance (Figure 1A).To test the osteogenic differentiation capacity, isolated cells were cultured in an osteogenic induction medium.SHED showed osteoblastlike morphology and after staining with Alizarin Red, calcium deposits were evidenced (Figure 1B).The expression of typical mesenchymal and embryonic marker genes in SHED was assessed using RT-PCR.SHED expressed not only the marker genes for MSC (NES and CD105) but also for embryonic cells (NANOG and OCT-4) (Figure 1C).The marker genes for hematopoietic lineages (CD34 and CD45) were not expressed, as expected. (12,55)he viability of SHED cultured in the presence or absence of agonist, antagonists, and TNF-α was not significantly different among the groups and when compared to that of cells cultured with the medium alone (data not shown).
with the group cultured only in the medium (Figure 2A).Thereafter, we analyzed whether TNF-α, agonist, or both could alter the levels of HLA-DR.Despite a slight increase in the level of this marker in SHED in the presence of TNF-α or TNF-α and agonist, there was no statistical difference between the groups (Figure 2B).
Subsequently, the effect of the antagonists was analyzed within the groups that received TNF-α, agonist, or their combination (Figure 2C to F).The presence of the antagonists alone did not lead to any change in HLA-DR levels (Figure 2C).However, the groups that received both the antagonists (Ant1+2) and also received the agonist (Figure 2D), TNF-α (Figure 2E) or Agonist+TNF-α (Figure 2F), showed a difference vis-à-vis their respective reference groups (Ago, TNF-α, and Ago+TNF-α).

HLA-DR expression
Initially, all groups were compared with the group cultured only with the medium (medium; without the presence of TNF-α, agonist, and antagonists) to ascertain the situation in which SHED would have altered amounts of HLA-DR on their surfaces.Only the groups that received TNF-α, agonist, and mainly antagonist 2 (Ago+Ant2+TNF and Ago+Ant1+2+TNF) treatment showed a statistically significant difference compared

PD-L1 expression
In the comparison of all groups with the Medium Group, the groups that received antagonist 2 or both the antagonists in the presence of TNF-α, but independent of the agonist, showed a significant difference (Figure 3A).When comparing the means between the Medium and Ago, TNF-α, and Ago+TNF-α Groups, the groups that received TNF-α showed an increase compared to the Medium and Ago Groups, but the differences were significant only between the Medium and Ago+TNF-α Groups (Figure 3B).The analysis of the role of the antagonists revealed that the groups receiving both the antagonists showed a significant difference relative to their respective reference groups-without agonist and without TNF-α (Figure 3C), with agonists (Figure 3D), with TNF-α (Figure 3E), or with agonists and TNF-α (Figure 3F).

PD-L2 expression
Similar to the levels of the other markers, PD-L2 levels were significantly increased in the groups that received antagonists 1, 2, or both, in the presence of TNF-α relative to the levels in the Medium Group; however, in the Ant1+TNF-α and Ago+Ant1+TNF-α groups, the increase in PD-L2 levels was not significant (Figure 4A).SHED showed increased PD-L2 levels in the presence of TNF-α, but the increase was significant when SHED were cultured in the presence of TNF-α and the agonist (Figure 4B).As for the analyses for the antagonists in cells that did or did not receive agonists and TNF-α, there were no significant changes in the expression of the PD-L2 marker between the groups (Figure 4C to F).  ❚ DISCUSSION In most studies, MSCs are considered potent inhibitors of the immune system.It is believed that such property of MSCs is not fixed or constitutive, but is influenced by the inflammatory environment.The immune system should act as a mediator, regulating not only the immunomodulatory properties of MSCs but also their proliferation and differentiation, thereby, actively influencing the process of damage recovery.Some inflammatory molecules, such as TNF-α and IFN-γ, appear to be involved in this process. (3,8,10,15,23,25)esenchymal stromal cells apparently have an immunoregulatory role and an antigen-presenting cell (APC) role, depending on the type, intensity, and timing of the inflammatory process.At the commencement of the inflammatory process, MSCs increase their antigenpresenting potential for fighting a possible infection.(58)(59)(60)(61)(62) Different types of MSCs share immunomodulatory effects and have great therapeutic potential.The immunomodulatory capacity of dental pulp stem cells (DPSCs) and SHED is no exception, and these cells sometimes show superior capacity compared with traditional bone marrow MSCs (BMSCs). (1,51,56)47) Molecules involved in the ECS are produced from membrane phospholipid precursors, and exerting immunomodulatory actions, such as decreased production of proinflammatory cytokines and modulation of T-helper (Th) 1 and Th2 (TH1/TH2) cell responsiveness. (39,42,63,64)esenchymal stromal cells can express all the components of the ECS.AEA, CB1, and CB2 are expressed and are involved in the physiological protection against intense inflammatory responses, with CB1 participating in the healing of inflamed tissues.Furthermore, CB1 is activated during osteogenic differentiation of BMSCs and periodontal ligament stem cells, even under an inflammatory environment.CB1 was reported to be an essential component in the survival of differentiated cells during acute stress. (48,49)he present study sought to evaluate the role of the ECS in the presence of TNF-α and to assess the possible cellular phenotypic alterations.In general, TNF-α and CB1 and CB2 receptor antagonists, especially the CB2 antagonist, were able to alter the immunological phenotype of the ECS with regard to the three markers analyzed.Because there was no significant difference in the cell viability test, any change occurring between the groups is expected to result from the alteration in the immunological phenotype of SHED.
We noted that not only the presence of TNF-α together with the antagonists (mainly of CB2) but the presence of the agonist also led to increased HLA-DR expression in SHED.In this regard, anandamide may be activating other ECS receptors, such as G protein-coupled receptor 55 (GPR55) and transient receptor potential (TRP) channels and their respective subfamilies. (43)These results indicate that SHED exhibit immunological response under these conditions.Palomares Cabeza et al. (34) showed that, under inflammatory conditions, these cells are able to interact with T lymphocytes via HLA-DR, because HLA-DR is especially responsible for initiating adaptive immunity by presenting antigens to these lymphocytes.The results of the present study are in agreement with the notion that SHED function as APCs or the with the possibility of inhibiting inflammatory processes via the presentation of specific antigens to T cells, depending on the degree of inflammation of the microenvironment.The presence of SHED in the inflammatory environment leads to decreased proliferation of Th cells and increased proliferation of Treg, (9,(56)(57)(58) explaining the targeting/ maintenance of an immunoregulatory profile.
From figures 3A and 4A, it is evident that TNF-α together with the antagonists could drive SHED toward an immunomodulatory profile both via PD-L1 and PD-L2.This result was most evident for PD-L1, in which case the blockade of CB1 and CB2 increased the levels of this marker, especially in the presence of TNF-α.(58) Specifically, the CB1 receptor antagonist showed a slight, but nonsignificant, increase in HLA-DR and PD-L1 levels when SHED were cultured in the presence of TNF-α.The contribution of the CB2 antagonist, in the presence of TNF-α, on all three markers was evident, most notably for HLA-DR and PD-L1.However, the changes were only significant when both the antagonists were used.In this sense, the literature regarding CB1 and CB2 receptors is contradictory, with the consensus being only on the participation of the ECS in immunomodulation, but not on the role in activation and blockade of each receptor. (39,42,63,64)Galve-Roperh et al. (6) reported that CB2 activation is associated with chronic inflammation of the nervous system as well as with different immune disorders.Montanari et al. (46) described a series of novel benzofuran-based compounds with neuroprotective and immunomodulatory properties for the treatment of Alzheimer's disease, resulting in the change of proinflammatory M1 phenotype to the neuroprotective M2 phenotype of microglia.Rossi et al. (50) reported that CB1 promotes inflammation while CB2 regulates its magnitude.Moreno et al. (42) noted that CB2 ligands are responsible for the observed immunomodulatory effects.
The ECS is present in both immune system cells and MSCs, expressing both CB1 and CB2 receptors, which is suggestive of a possible common route of immunomodulation, mainly via CB2. (48,50,64)In a study with endothelial progenitor cells, the release of AEA and 2-AG was verified.In the presence of TNF-α, there was an increase in 2-AG levels.Moreover, after treatment of this mature cell line with endocannabinoids, a reduction in the induction (by TNF-α) of the proinflammatory adhesion molecule CD106 (VCAM) was observed. (65)n addition, cannabidiol was also reported to protect oligodendrocyte progenitor cells from apoptosis induced by lipopolysaccharides (LPS)-or INF-γ-stimulated inflammation.Cannabidiol treatment reversed the induction of caspase 3, decreased the production of reactive oxygen species and endoplasmic reticulum stress-induced apoptosis, followed by decreased levels of molecular effectors, Bax and caspase 12. (39) The expression of CB1 receptor is increased during osteogenic differentiation of MSCs and it is essential for the survival of these differentiated cells. (48)etrahydrocannabinol (THC) was able to activate the CB2 receptor, increasing BMSC immunoregulation of the release of inflammation-associated cytokines (TNF-α, interleukin [IL]-1β, IL-6, and IL-8).Furthermore, hyperalgesia and allodynia were significantly reduced when MSCs pretreated with THC were administered; consequently, the levels of proinflammatory cytokines were also significantly reduced, whereas IL-10 levels were increased. (66)It was shown that CB2 activation in MSCs led to increased IL-10 release and reduced levels of LPS-induced proinflammatory cytokines, IL-1β, IL-8, and IL-17. (50)n a study analyzing the molecular phenotype of gingival mesenchymal stromal cells (GMSCs), treatment with cannabidiol was verified to prevent the expression of genes of the NALP3-inflammasome pathway, suppressing the levels of NALP3, CASP1, and IL-18, demonstrating the inhibition of apoptosis, accompanied by the suppression of Bax.Cannabidiol treatment could also reduce the expression of genes involved in the activation of the immune system (CD109, CD151, CD40, CD46, CD59, CD68, CD81, CD82, CD99), while stimulating the expression of genes involved in the inhibition of immune responses (CD47, CD55, CD276) Libro et al. (67) In another study, pretreatment of GMSCs with cannabidiol led to the attenuation of the expression of genes implicated in the etiopathogenesis of Alzheimer's disease, showing that preconditioned GMSCs have potential for the treatment of early-stage Alzheimer's disease. (67)8) ❚ CONCLUSION The inhibition of the endocannabinoid receptors together with the proinflammatory effect of TNF-α led to an increased expression of HLA-DR on the surface of stem cells from human exfoliated deciduous teeth, and also conferred an anti-inflammatory profile on these cells by increasing the PD-L1 and PD-L2 levels.The agonist treatment also influenced the alteration of these surface proteins; however, we could not show the exact modulation of the immune profile of stem cells from human exfoliated deciduous teeth because the endocannabinoid system is a complex system and involves other receptors that were not analyzed in this study.Nonetheless, this study contributes to broadening the investigation of the interaction of stem cells from human exfoliated deciduous teeth and the endocannabinoid system in relation to immunomodulation.The interactions among the mesenchymal stromal cells, immune system, and the endocannabinoid system revealed in this study highlight the importance of investigating the cellular and physiological effects of active principles that act on the endocannabinoid system for the elucidation and demystification of their actions and consequences in the treatment of diseases.

Figure 2 .Figure 1 .
Figure 2. Median fluorescence intensity of human leukocyte antigen-DR isotype (HLA-DR) on the surface of stem cells from human exfoliated deciduous teeth (SHED).A) All groups compared with medium; B) SHED treated with tumor necrosis factor alpha (TNF-α), agonist, or both, or left untreated; C) SHED treated with antagonist 1, 2, or both, or left untreated; D) SHED in the presence of the agonist and treated with antagonist 1, 2, or both, or left untreated; E) SHED in the presence of TNF-α and treated with antagonist 1, 2, or both, or left untreated; F) SHED in the presence of the agonist and TNF-α and treated with antagonist 1, 2, or both, or left untreated.a -groups showing statistical difference at p<0.05 in relation to the Medium Group; b -groups showing statistical difference at p<0.01 in relation to the Medium Group

Figure 3 .
Figure 3. Median fluorescence intensity of programmed death ligand 1 (PD-L1) on the surface of stem cells from human exfoliated deciduous teeth (SHED).A) All groups compared with Medium; B) SHED treated with tumor necrosis factor alpha (TNF-α), agonist, or both, or left untreated; C) SHED treated with antagonist 1, 2, or both, or left untreated; D) SHED in the presence of the agonist and treated with antagonist 1, 2, or both, or left untreated; E) SHED in the presence of TNF-α and treated with antagonist 1, 2, or both, or left untreated; F) SHED in the presence of the agonist and TNF-α and treated with antagonist 1, 2, or both, or left untreated.a -groups showing statistical difference at p<0.05 in relation to the Medium Group; b -groups showing statistical difference at p<0.01 in relation to the Medium Group

Figure 4 .
Figure 4. Median fluorescence intensity of programmed death ligand 2 (PD-L2) on the surface of stem cells from human exfoliated deciduous teeth (SHED).A) All groups compared with Medium; B) SHED treated with tumor necrosis factor alpha (TNF-α), agonist, or both, or left untreated; C) SHED treated with antagonist 1, 2, or both, or left untreated; D) SHED in the presence of the agonist and treated with antagonist 1, 2, or both, or left untreated; E) SHED in the presence of TNF-α and treated with antagonist 1, 2, or both, or left untreated; F) SHED in the presence of the agonist and TNF-α and treated with antagonist 1, 2, or both, or left untreated.a -groups showing statistical difference at p<0.05 in relation to the Medium Group; b -groups showing statistical difference at p<0.01 in relation to the Medium Group

Table 2 .
Description of the groups used for immunomodulation of stem cells from human exfoliated deciduous teeth einstein (São Paulo).2023;21:1-9